In Vitro Differentiation of Human Umbilical Cord Blood CD133(+)Cells into Insulin Producing Cells in Co-Culture with Rat Pancreatic Mesenchymal Stem Cells.

OBJECTIVE
Pancreatic stroma plays an important role in the induction of pancreatic cells by the use of close range signaling. In this respect, we presume that pancreatic mesenchymal cells (PMCs) as a fundamental factor of the stromal niche may have an effective role in differentiation of umbilical cord blood cluster of differentiation 133(+) (UCB-CD133(+)) cells into newly-formed β-cells in vitro.


MATERIALS AND METHODS
This study is an experimental research. The UCB-CD133(+)cells were purified by magnetic activated cell sorting (MACS) and differentiated into insulin producing cells (IPCs) in co-culture, both directly and indirectly with rat PMCs. Immunocytochemistry and enzyme linked immune sorbent assay (ELISA) were used to determine expression and production of insulin and C-peptide at the protein level.


RESULTS
Our results demonstrated that UCB-CD133(+)differentiated into IPCs. Cells in islet-like clusters with (out) co-cultured with rat pancreatic stromal cells produced insulin and C-peptide and released them into the culture medium at the end of the induction protocol. However they did not respond well to glucose challenges.


CONCLUSION
Rat PMCs possibly affect differentiation of UCB-CD133(+)cells into IPCs by increasing the number of immature β-cells.


Introduction
Approximately 200 million people worldwide suffer from diabetes mellitus. This figure may double by 2025. The common treatment for type1 diabetes is daily injections of insulin (1). Transplantation of pancreatic islets is another method to treat diabetes, however worldwide shortage of transplant-ready islets, immune rejection and recurrent attacks against islets by underlying autoimmunity have yet to be resolved (2,3). Application of in-sulin producing cells (IPCs) derived from adult stem cells is a potentially attractive strategy and may serve as a means to overcome many of the major issues that can complicate cell-based therapies, such as immune rejection and shortage of suitable donors (1). To this end, mesenchymal and hematopoietic stem cells from bone marrow and umbilical cord blood (UCB) represent valuable sources for differentiation into pancreatic β cells (4).
Many studies have reported that the microenvironment surrounding stem cells controls differentiation in vivo (5,6). To our knowledge, stromal niches can be thought of as discrete anatomical sites that contain niche support cells which physically contact adjacent cells and influence stem cell behavior via close range signaling (6). It has been reported that pancreatic mesenchyme controls the timing of β-cell differentiation by secreted soluble factors. However, the identity of the soluble factors and mechanisms involved in regulating the development and function of the pancreas remains unknown (7). Other studies have reported that extract of rat pancreas could induce mesenchymal stem cell (MSC) differentiation into IPCs in vitro with concomitant increases of insulin. However the extract could not induce functionally mature pancreatic cells responsive to different concentrations of glucose (8)(9)(10).
Therefore, the purpose of our study was to investigate the differentiation of human UCB-cluster of differentiation 133 + (CD133 + ) cells into IPCs in co-culture with rat pancreatic MSCs (PMCs).

Isolation and culture of umbilical cord blood cluster of differentiation 133 + cells
This study is an experimental research. Fresh cord blood samples obtained from the Royan Public Cord Blood Bank were immediately diluted with HAES-Steril (Free flex, Germany) 10% at 1:5 (v/v) to accelerate red blood cell (RBC) sedimentation and facilitate isolation of cord blood mononuclear cells (MNCs). Subsequently, the MNCs were isolated using a ficoll density gradient (Inno-Train, Germany) and then washed twice in phosphate buffer saline (PBS, Invitrogen, USA) that contained 0.5% fetal bovine serum (FBS, Sigma, USA) and 2 mM ethylenediaminetetraacetic acid (EDTA, sigma, USA). Magnetic cell sorting (MACS, Milteny Biotech, Bergisch Gladbach, Germany) was used for isolation of CD133 + cells according to the manufacturer's guidelines. Briefly, 100 μL of FcR blocking and 100 μL of CD133 microbeads were added to at least 1×10 8 MNCs/300 μL, then mixed and incubated for 30 minutes at 2-8˚C. After washing with PBS that contained 0.5% FBS and 2 mM EDTA, cells were resuspended in 500 μL of the same PBS solution. A MACS column was used to isolate highly pure CD133 + cells from the cell suspension according to a data sheet. A sample fraction of the purified cells was checked for viability, cell number, morphology and purity.

Isolation and culture of rat pancreatic mesenchymal stem cells
We isolated rat PMCs by removing the pancreases of 7-day postnatal Wistar rats (n=5) according to a protocol approved by the Institutional Review Board and Institutional Ethical Committee at Royan Institute. Briefly, pancreas tissue was diced into 1 mm³ pieces in RPMI 1640 that contained 1 mg/ml collagenase type 1a (Sigma, Germany) using sterile blades and incubated for 90 minutes at 37˚C. The collagenase solution was inactivated with RPMI 1640 supplemented with 15% FBS. Cell clumps and undissociated tissue were removed by passing the tissue through a nylon mesh filter (100 mm). Washed cells were resuspended in RPMI 1640 (Sigma, Germany) supplemented with 10% FBS, 100 IU/ml penicillin (Invitrogen, Germany), 100 mg/ml streptomycin (Invitrogen, USA) and 2 mM L-glutamine (Invitrogen, USA). Cells were then seeded in 25 cm 2 culture flasks (Cellstar, Greiner, Germany). Two days later, the medium was changed to remove non-adherent cells. When cells reached proper confluency they were trypsinized (5 mg trypsin/ml PBS), washed, resuspended in 20 ml medium and cultured in 75 cm 2 flasks.

Osteogenic and adipogenic differentiation
Rat PMCs at passage three were used for osteogenic and adipogenic differentiation. Rat PMCs were cultured for 21 days in Dulbecco's Modified Eagle Medium (DMEM) that contained 10% FBS, 50 mg/ml ascorbic acid 2-phosphate, 10 nM dexamethasone and 10 mM b-glycerol phosphate (all purchased from Sigma, Germany). Differentiation was confirmed by observation of extracellular matrix calcification using alizarin red staining.
60 mM indomethacin, 10 nM dexamethasone and 10 mg/ml acid ascorbic (all from Sigma, USA) was used as the differentiation medium for adipogenic differentiation. Passage-3 rat PMCs were used for these experiments. Differentiation media were changed every 3 days; after 21 days, cells were fixed with cold 10% formalin (sigma, Germany) for 1 hour, then washed with water and stained with oil-red solution (Sigma, Germany) for 2 hours at room temperature. The presence of intra-cellular lipid droplets in the cytoplasm was observed with an optical microscope. Figure 1 illustrates the protocol to induce β-cell differentiation in the presence or absence of PMCs. Briefly, 1×10 6 CD133 + cells were plated into six-well plates in 10% FBS in DMEM according to the protocol by Shi et al. (11). Subsequently, 100 ng/ml activin A (Sigma, Germany) was added and cells were allowed to incubate at 37˚C, 5% CO 2 and 95% humidity for 24 hours. At the end of the incubation time, the medium was changed to DMEM that included 10% FBS for 6-8 hours followed by addition of 1 µM all-trans retinoic acid (Sigma, Germany) for an extra 24 hours (step 1). The medium was changed to DMEM supplemented with 10% FBS and 10 ng/ml of basic fibroblast growth factor (bFGF, Sigma, Germany) for 3-5 days (step 2). Finally, cells were cultured in DMEM/F12 with N2 and B27 supplements (all from Gibco-BRL-USA), 1 μg/ml laminin (Sigma, Germany), 10 ng/ ml bFGF and 10 mm nicotinamide (Sigma, Germany) for an additional 3-5 days (step 3).

Co-culture of CD133 + cells with rat pancreatic mesenchymal stem cells
To investigate the effect of rat PMCs on differentiation of UCB-CD133 + into IPCs, expanded rat PMCs at passage-3 were plated in six-well plates and allowed to grow to approximately 80-90% confluency. Mitomycin C (Sigma-Aldrich, USA), at a concentration of 25 μg/ml, was subsequently added to the culture medium to prevent stromal cell proliferation and the cells were incubated at 37˚C, 5% CO 2 and 95% humidity overnight. The next day, UCB-CD133 + cells were directly seeded on the inactivated stromal cell layer at a ratio of (1:1) or on the transwell system (Fig.1D). Then, the differentiation protocol was performed according to the Shi et al. (11) method.

Immunocytochemistry
Differentiated cells were fixed with 4% paraformaldehyde for 20 minutes at 4˚C, washed several times and blocked for 30 minutes in 10% goat serum (Gibco, USA). Next, cells were allowed to incubate overnight with mouse anti-human insulin 1:500 (Sigma-Aldrich, USA) and mouse anti-human C-peptide 1:250 (Abcam, Cambridge, UK) as primary antibodies at 4˚C, followed 3 times washing with PBS. FITC-conjugated goat anti-mouse antibody (1:100, Sigma, Germany) was applied for 1 hour at 37˚C and subsequently washed by PBS to remove unwanted conjugated antibodies. Nuclei were counterstained with 4΄,6-diamidino-2-phenylindole (DAPI) and cells were visualized using a fluorescence microscope (Olympus BX51, Japan).

Enzyme-linked immunosorbent assay (ELISA)
In order to test whether the insulin released from differentiated cells was glucose-dependent, we used two glucose concentrations (2.5 mM and 27.5 mM) (12). The human insulin and C-peptide levels in culture supernatants were measured by a Human Insulin ELISA Kit and Human C-peptide ELISA Kit (Mercodia, Switzerland) according to the manufacturer's instructions. The differentiated cells were pre-incubated in Krebs-Ringer buffer (120 mM NaCl, 5 mM KCl, 2.5 mM CaCl 2 , 1.1 mM MgCl 2 , 25 mM NaHCO 3 and 0.1% bovine serum albumin) at 37˚C for 90 minutes. Then, Krebs-Ringer buffer (Sigma, Germany) was re-placed by Krebs-Ringer buffer that contained 5 mM glucose at 37˚C for 15 minutes in order to determine the basal level of insulin and Cpeptide. To induce the release of insulin, the same differentiated cells were subsequently incubated with 27.5 mM glucose for an additional 15 minutes.

Reverse transcription polymerase chain reaction (RT-PCR) analysis
The expressions of CD133, OCT4 and NANOG were analyzed by RT-PCR. Briefly, total RNA was extracted from cells using RNX reagent (Sinagene, Iran) according to the manufacturer's instructions. cDNA was generated by a cDNA Synthesis Kit (Fermentas, USA). cDNA was subjected to RT-PCR using specific primers for CD133, OCT4 and NA-NOG (Table 1). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. PCR conditions were: 94˚C for 4 minutes, 30 cycles of 94˚C for 1 minute, 60˚C for 45 seconds, 72˚C for 1 minute, and 72˚C for 10 minutes. PCR products were separated on a 2% agarose gel that contained ethidium bromide (Invitrogen, USA) and photographed.

Real-time quantitative polymerase chain reaction (qRT-PCR)
The expressions of GLUCAGON, INSULIN, PDX1 and NKX6.1 were analyzed by qRT-PCR. Briefly, cells were harvested from differentiating plates and the total RNA was isolated from triplicate samples by an RNA Extraction Kit (TaKaRa, Japan). We used 100-500 ng for reverse transcription with a Prime Script II Strand cDNA Synthesis Kit (TaKaRa, Japan). PCR reactions were run in duplicate using 1/40 th of the cDNA per reaction and 400 nM forward and reverse primers with SYBR Green Master Mix (TaKaRa, Japan). RT-PCR was performed using the Rotor Gene 3000 (Corbett Research, Germany). QRT-PCRs were performed in duplicate for each sample primer set, and the mean of the three experiments was used as the relative quantification value. Relative gene expression was analyzed using the comparative C t method, 2 -ΔΔCt . All samples were normalized to the levels of GAPDH, which was used as the loading control. Primer sequences related to specific pancreatic genes are listed in table 1.

Statistical analysis
The data are presented as mean ± standard error (SE). Each experiment was repeated in triplicate. Statistical significance was determined using the Univariate analysis of variance, two-way ANOVA and repeated measures test. We used the nonparametric Mann-Whitney test to compare the two differentiation groups (with and without co-culture). P values less than 0.05 were considered statistically significant. Figure 1 is a schematic diagram for isolation and differentiation of UCB-CD133 + and rat MSCs. CD133 + cells were isolated using MACS tech-nology according to a Miltenyi Biotech protocol. The purified cells were passed twice through the MACS column to increase cell purity. The percentage of cells prior to purification was 0.1-0.4%, and reach to approximately 91%, after purification. Purified cells expressed strong bands of CD133, OCT4 and NANOG compared to cord blood MNCs when detected by RT-PCR ( Fig.2A, B).

Human umbilical cord blood cluster of differentiation 133 + cells and rat mesenchymal stem cells isolation and characterization
Adherent cells derived from the pancreases of 7-day postnatal Wistar rats also showed typical fibroblast-like structure that rapidly reached confluence (7-10 days). These cells showed a similar phenotype and expression pattern to Bone Marrow-MSCs. They were positive for CD44 and CD90, negative for CD45 and CD11b (Fig.2C). The cells differentiated into osteocytes and adipocytes in differentiation medium (Fig.2D).

Cell differentiation
Differentiation into IPCs was performed according to the Shi et al. (11) protocol (Fig.1D). The protocol included cell expansion and production of forgut endoderm cells by addition of activin A, followed by treatment with all-trans retinoic acid in the presence of serum, differentiation into posterior like cells in presence of bFGF and finally, differentiation into pancreas progenitor cells with laminin and nicotinamide.
Throughout induction of cells with the Shi et al. (11) protocol, a number of changes appeared in cell morphology. The CD133 + cells expanded in the first step; in the second step, dense colonies formed which were positive for C-peptide and insulin in the final step (Fig.3A). Untreated CD133 + cells and PMCs were used as negative controls (Fig.3B).

Effect of rat pancreatic mesenchymal stem cells on differentiation into insulin producing cells
In order to determine whether PMCs, as an effective factor in the pancreas niche, could increase efficiency of IPCs production, we directly and indirectly co-cultured the cells with rat PMCs (Fig.1C).
As shown in figure 4, the number of colonies was higher when UCB-CD133 + cells had cellto-cell contact with rat PMCs compared to the indirect culture. The cells in both groups were positive for C-peptide and insulin when stained with monoclonal antibodies against these proteins. No significant difference could be observed in the expression of pancreatic specific genes INSULIN, GLUCAGON, NKX6.1, and  PDX1 in the presence or absence of rat PMCs at ten days post-induction. The differentiated cells from UCB-CD133 + cells did not secret valuable levels of insulin and C-peptide in the supernatant tested by ELISA (Fig.5) and in response to different concentrations of glucose. Unexpectedly, the highest levels of insulin secretion were observed in response to the supernatants that contained 5 μM of glucose (P<0.05). The undifferentiated cells (negative control) showed no significant release of insulin into the medium (data not shown).

Discussion
The possibility of generating IPCs from various cellular sources including embryonic stem cells (11)(12)(13)(14)(15) and MSCs (16)(17)(18)(19)(20)(21) has been recently demonstrated. However, the efficiency of differentiation and the normal function of these newly formed β-cells, especially in response to glucose stimulation are challengeable questions. Stromal niches can be thought of as discrete anatomical sites that contain niche support cells which physically contact adjacent cells and influence stem cell behavior via close range signaling (6). In this study we have differentiated UCB-CD133 + cells into IPCs both with and without rat PMCs in an attempt to investigate the effect of the pancreatic stromal niche on generating IPCs.
In the present study, we generated IPCs through a three step protocol and confirmed the presence of insulin production by immunofluorescence. The functionality of the in vitro generated IPCs was tested by measuring insulin and C-peptide release in response to glucose challenges.
Our results demonstrated that purified CD133 + cells expressed CD133, OCT4 and NANOG genes which confirmed their stem potential. They easily formed islet-like colonies at the end of the differentiation process. The colonies expressed insulin and C-peptide at the protein level but were not functional and could not increase insulin production and release in response to high glucose concentrations.
We investigated the effect of the pancreatic stromal niche on generating IPCs from UCB-CD133 + cells by using a co-cultured system. Our finding showed no significant differences between cells cultured in the presence or absence of rat PMCs, however in direct culture the number of colonies increased. Other studies have also confirmed that rat pancreas extract could stimulate phenotypic pancreatic differentiation (8,9). As our differentiated cells did not significantly express specific protein markers (insulin and C-peptide) and were unresponsive to high glucose concentrations, thus we proposed that pancreatic stromal cells might not be sufficient to induce functionally mature pancreatic differentiated cells as previously reported by Lumelsky et al. (22).
Apoptotic cells can take up exogenous insulin from culture medium. We have measured insulin and C-peptide protein to demonstrate de novo synthesis of insulin (12). We did not detect C-peptide in the pancreatic differentiation medium before differentiation (day 0, data not shown). Therefore, detectable levels of C-peptide in culture supernatants collected after differentiation should come from differentiated cells. Both differentiated cells (with and without co-culture) secreted insulin and C-peptide in response to glucose stimulations. However, there was no significant difference in secretion of insulin and C-peptide between these two groups. Glucose stimulation assays showed that IPCs generated by these groups released insulin and C-peptide in response to minimal glucose (5 μM) stimulation, yet did not release significant amounts of insulin and C-peptide at a higher glucose (27.5 μM) stimulation. IPCs might therefore merely release all of the insulin and C-peptide after stimulation with a low dose of glucose and have no insulin reserve to release upon re-stimulation at a higher dose of glucose. The time taken by IPCs to synthesize pro-insulin and process insulin might exceed the one hour time span in which the cells were incubated with the higher dose of glucose. Thus, IPCs could have released all of their intracellular insulin and had insufficient time to resynthesize more insulin for release during the incubation period at the higher glucose level. On the other hand, IPCs might not have been mature enough; therefore, the number of glucose receptors was not sufficient, which caused them to lack normal function in response to glucose stimulations. Additional, more extensive studies would be necessary to clarify the reasons for these observations.

Conclusion
The data presented in this study indicated that human UCB-CD133 + cells could differentiate into IPCs in vitro. Pancreatic stromal cells might cause an enhancement in the number of immature pancreatic β-cells, but not in the number of mature cells. More research would be necessary to determine the role of other factors of the pancreatic niche on the differentiation of β-cells in generating IPCs in vitro.
their appreciation to Dr. Hamid Gourabi, Dr. Abdolhossein Shahverdi and Dr. Ahmad Vosough for providing the research grant and for their supportive assistance during this research. Human cord blood was obtained from Royan public Cord Blood Bank. The authors declare that they have no conflict of interests.